University of Groningen Total synthesis of mycolic acids and site-selective functionalization of aminoglycoside antibiotics Tahiri, Nabil

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University of Groningen

Total synthesis of mycolic acids and site-selective functionalization of aminoglycoside

antibiotics

Tahiri, Nabil

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Tahiri, N. (2019). Total synthesis of mycolic acids and site-selective functionalization of aminoglycoside antibiotics. University of Groningen.

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Chapter 2:

Synthesis of the Methoxymycolic Acid

Fragments

Part of this chapter will be submitted for publication:

N. Tahiri, P. Fodran, D. Jayaraman, J. Buter, T. A. Ocampo, I. Van Rhijn, D. B. Moody, M. D. Witte, A. J. Minnaard, manuscript in preparation

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2.1 Introduction

Although the stereochemistry in the α-alkyl β-hydroxy segment seems established to be

R,R, by optical rotation[1]_{and in vitro testing of natural 2R,3R and racemic glucose}

monomycolate stereoisomers,[2]_{the remaining functionalities, in particular the}

cyclopropyl moiety, seem yet to be defined regarding their absolute stereochemistry. Therefore, four diastereomers of methoxymycolic acid were synthesized with all possible diastereomers in the syn α-methyl methoxy and cis cyclopropyl functionalities. This chapter describes the synthesis of all three fragments required for the convergent total synthesis of this mycolic acid.

In order to allow the efficient synthesis of all four desired methoxymycolic acid diastereomers, we envisioned that a convergent synthesis would be the most optimal approach. Dividing the molecule in three parts of roughly equal stereochemical complexity would allow construction of any diastereomer, by proper combination of the individual fragments A, B and C (Scheme 1). The specific disconnections between fragment A and B, and fragment B and C, were based on the fact that cis-configured cyclopropyl moieties are readily introduced on allylic alcohols via Charettes method, and that the required allylic alcohol can be prepared from readily available building blocks (for detailed description see section 2.3). The choice for the specific disconnections was further based on the availability of commercial starting materials that are required for the introduction of the other stereocenters and correct linker lengths. In order to vary the stereochemistry in the α-methyl methoxy and cyclopropyl moieties, both enantiomers of fragments B and C were synthesized whereas fragment A was synthesized only as the R,R isomer.

Scheme 1. Retrosynthetic analysis of mycolic acid.

Combining the fragments by utilizing a sp3_-sp3 _{cross-coupling such as the Suzuki-Fu}

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required. We therefore applied this chemistry for the combination of fragments B and

C. However, disconnecting next to the cyclopropyl moiety excludes the application of a

Suzuki-Fu coupling, since insertion of palladium will result in cyclopropyl cleavage. We therefore turned to a modified Julia-Kocienski olefination, which requires an additional reduction step of the newly formed double bond.

2.2 Fragment A

2.2.1 Retrosynthetic analysis

The most challenging aspect in the synthesis of fragment A is the construction of the α-alkyl, β-hydroxyl acid moiety. Intuitively, construction of both stereocenters seemed to be best achieved by means of an aldol reaction. Although the body of literature on the asymmetric aldol reaction is huge, methods on its asymmetric anti-selective segment is much less represented. From the available options, the methodology developed by Masamune[3] _{seemed to be matching best with our desires (Scheme 2): easy access to}

the auxiliary and high stereoselectivity with linear aliphatic aldehydes. Therefore, fragment A is best represented as intermediate 1 which contains an ephedrine based auxiliary, readily installed by esterification, to allow for the aldol reaction to proceed with high stereocontrol. Moreover, this auxiliary can be maintained throughout the synthesis in order to provide enhanced solubility while also acting as a protection group for the carboxylic acid functionality. Disconnection between the α- and β-carbons of the ester, yields aldehyde 5 (which had to be prepared), and further installment of the α-branch can be achieved by means of chain elongation of the bromoester of 3 and 2 with 1-hexadecene (4) using a Suzuki-Fu cross-coupling. Compounds 3 and 4 are low-cost commercially available materials, whereas 2 is affordable.

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24 2.2.2 Synthesis of the aldehyde spacer

The synthesis of fragment A started with the synthesis of linker 5, which was required for the aldol reaction. Commercially available pentadecanolide (6) was reduced cleanly using DIBAL to its lactol counterpart (Scheme 3). On small scale the workup was eventless, but on a 14 gram scale excessive extractions with DCM were required in order to achieve decent recovery of the product due to its low solubility. Lactol 7 was subjected to a Horner-Wadsworth-Emmons reaction,[4]_{resulting in the α,β-unsaturated}

thioester 8 in 70% overall yield over two steps. In the first instance, we planned a step-by-step conversion of 8 to 5 by means of hydrogenation of the double bond, silyl protection of the alcohol and subsequent DIBAL reduction of the thioester to the aldehyde. However, because hydrogenation over Pd/C was unsuccessful, probably because of traces of thiols resulting in catalyst poisoning, we realized that a Fukuyama reduction might actually achieve all three conversions in one single step. Indeed, treatment of 8 with triethylsilane in acetone in the presence of Pd/C resulted in the hydrogenated TES protected aldehyde 5 in excellent purity after column chromatography. Although three transformations were in this way efficiently achieved in one step, the yield turned out to differ considerably from batch to batch. In this reaction, seven intermediates can be formed varying considerably in polarity, and this might suggest full conversion of the starting material while actually the reaction is not complete. Furthermore, yields were generally lower when the reaction was performed on multi-gram scale compared to sub-gram scale.

Scheme 3. Synthesis of aldehyde 5 required for the aldol reaction.

Since later in the Masamune aldol reaction the TES protection group proved to be too labile, we opted for the installment of a more stable protection (such as a TBS group) at this stage. Therefore, instead of TESH, TBSH was used in the Fukuyama reduction (Scheme 4). Unfortunately, no noticeable conversion of 8 was observed by TLC. Alternatively, the Fukuyama reduction of TBS protected thioester 10 could provide a quick entry to TBS protected aldehyde 5b. TBS protection of 8 under standard conditions went smoothly in quantitative yield, but surprisingly, Fukuyama reduction of

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Scheme 4. Attempted synthesis of aldehyde 5b.

In an attempt to obtain TBS protected aldehyde 5b, we revisited the step-by-step approach in which first the thioester 10 was reduced to the alcohol, followed by hydrogenation of the double bond, and subsequent oxidation of the alcohol to the aldehyde (Scheme 5). Reduction of TBS thioester 10 with LiAlH4 went in quantitative

yield, but resulted in an inseparable mixture of saturated alcohol 11 and allylic alcohol

12 in a 2:1 ratio, respectively. Reduction of the double bond with Pd/C under H2

atmosphere was unsuccessful, and reduction using our in-house developed diimide reduction[5]_{resulted in complete hydrogenation, but concomitant cleavage of the TBS}

group led to the corresponding diol. We therefore decided to continue with the Fukuyama reduction as depicted in Scheme 3 and address the problems associated with the TES group at a later stage in the synthesis.

Scheme 5. Attempted synthesis of 11 from 10.

2.2.3 Completion of fragment A

With aldehyde 5 in hand, the synthesis of fragment A continued by the in situ generation of the acyl bromide of 3 using oxalyl bromide, and subsequent esterification with 2 in quantitative yield. This was to prevent scrambling of the primary bromide with chloride, which occurs when preparing the acid chloride using oxalyl chloride. Masamune aldol[3]_{reaction of chiral ester 13 with aldehyde 5 yielded the desired}

β-hydroxy ester in a 9:1 diastereomeric ratio. Unfortunately, upon workup the TES group was partially cleaved, and it was therefore decided to completely remove the TES group by stirring the crude reaction mixture in acidic THF. Although no separation of the diastereomers was observed on TLC, a substantial separation was achieved by applying a slow gradient on column chromatography. By careful analysis of the collected fractions with 1_{H-NMR, and by combining all fractions with a dr better than 95:5, the}

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very high dr of 97:3. Although this was a very laborious and time consuming process, scaling up the reaction to 10 g of chiral ester did not cause any problems and resulted in more than 8 g of diol 14. This amount was sufficient to complete the synthesis of all four diastereomers. The remaining steps in the synthesis of fragment A proceeded with relative ease, and consisted of TBS protection of both alcohols in 14, resulting in compound 15 in good yield. Compound 16 was obtained by chain elongation of 15, by applying a sp3_-sp3_Suzuki-Fu[6]_{cross-coupling. Then, the selective deprotection of the}

primary alcohol went in excellent yield, resulting in 17. We decided to perform this deprotection under acidic conditions to prevent any possible loss of the β-silylether via an E1cb elimination. Finally, alcohol 17 was oxidized in high yield to aldehyde 1 by applying a Dess-Martin periodinane oxidation. The crude aldehyde could be purified by multiple acetonitrile/pentane extractions, followed by a simple column purification. This aldehyde was stored under nitrogen at -20 °C for at least two months without any noticeable degradation. Overall, fragment A (1) was synthesized in nine steps (longest linear sequence) from commercially available pentadecanolide (6) in 24% overall yield and 97:3 dr.

Scheme 6. Completion of fragment A.

2.3 Fragment B

Fragment B, the middle segment of the molecule containing the cis cyclopropyl functionality, can be derived from alcohol 18 after a few simple functional group interconversions (Scheme 7). The alcohol 18 can be obtained from allylic alcohol 19 by

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means of a Charette asymmetric cyclopropanation.[7,8]_{Since the stereochemistry in 19 is}

transferred to the cyclopropanation product 18, it is of utmost importance to perform the cyclopropanation with an extremely high cis purity in 19, the key intermediate in the synthesis of fragment B. The allylic alcohol is necessary for coordination with the chiral promotor in order to establish efficient face discrimination in the cyclopropanation reaction. Because the synthesis of the cis alkene was challenging, we aimed to synthesize this compound via a Z-selective Horner-Wadsworth-Emmons reaction (Scheme 7, Route 1) of aldehyde 21, or by addition of 22 (Route 2) or 23 (Route 3) to paraformaldehyde.

Scheme 7. Retrosynthetic analysis of fragment B.

2.3.2 Route 1

In order to perform the modified Horner-Wardsworth-Emmons reaction of 21 and phosphonate 27, the starting materials had to be synthesized first. Phosphonate 27 was prepared on a 35 gram scale following a straightforward procedure by Touchard[9]

which started by the reaction of ethyl dichlorophosphite and 2-tert-butylphenol, followed by an Arbusov reaction with ethyl bromoacetate (Scheme 8). Although the procedure reported that the product of the Arbusov reaction could be used without any purification, we noticed that a simple trituration of the crude material in heptane resulted in analytically pure material in excellent yield. Because of the big scale, we decided to obtain aldehyde 21 using a Swern oxidation, and oxidation of 21 gram of alcohol 28 went smoothly. Next, Horner-Wardsworth-Emmons reaction of aldehyde 21 and phosphonate 27 at ˗78 °C, resulted in the α,β-unsaturated ester as a single isomer in 90% yield. In order to prevent any conjugate reduction, we subjected 29 to a DIBAL

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28

reduction. Unfortunately, close inspection of the 1_{H NMR spectrum}[10]_{revealed that up}

to 10% isomerization towards the trans isomer had occurred.[11]_{Cyclopropanation of}

this mixture, and comparison of the NMR spectrum with literature values for trans cyclopropyl compounds[12]_{further confirmed this observation, and clearly indicated}

formation of a trans cyclopropyl group. All attempts to separate the cis/trans isomers in

19 over silica and silver nitrate impregnated silica were unsuccessful, and therefore this

route was abandoned.

Scheme 8. Synthesis of key intermediate 19 (Route 1).

2.3.3 Route 2

Our second attempt to prepare intermediate 19 started with the alkyne zipper reaction[13]

of commercially available 30, followed by TBDPS protection of the crude, resulting in protected terminal alkyne 23 in 86% yield over two steps. Anti hydrometalation[14]_with

in situ generated HInCl2 of 23, followed by trapping with iodine resulted in cis vinyl

iodide 31 as a single isomer in moderate yield. Unfortunately, all attempts to obtain allylic alcohol 19 via transmetalation of 31 with BuLi, or formation of the corresponding Grignard reagent under standard conditions, followed by addition to paraformaldehyde, were unsuccessful.

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29 2.3.4 Route 3 and completion of fragment B

Since the conjugate base of an alkyne is significantly more stable compared to an alkene, we reasoned that the addition of a deprotonated alkyne to paraformaldehyde might be a more reasonable approach (Scheme 10). A subsequent stereospecific hydrogenation could lead to key intermediate 19. Following a literature procedure on a very similar substrate[15]_{resulted in the formation of 20 in good yield. Even though}

Lindlar reductions of substrates similar to 20 have been reported in literature,[16]_{and the}

method is considered a prototype of a syn-specific hydrogenation, in our hands a 10% isomerization towards the more stable trans isomer was observed. Fortunately, subjecting propargylic alcohol 20 to a nickel boride reduction,[17]_{originally reported by}

Brown and coworkers, resulted in spot to spot conversion on TLC. The NMR of the crude product seemed extremely pure at first glance, but after closer analysis, it was noticed that the integration of the olefinic signals did not match with the rest of the signals. Prolonging the relaxation time between scans to 10 s improved this ratio, but still did not led to a matching integration. Although no additional signals were observed on 1_{H-NMR, we suspected that most probably over-reduction was responsible for the}

imbalance in the integration. Comparison with literature 1_{H- and}13_{C-NMR spectra}

confirmed our suspicion and revealed that indeed the signals of the corresponding alkane[18]_{overlapped with the signals of our desired product 19. To our great}

satisfaction, this time the alkane side product and 19 were separable (to some extend) using a 20% AgNO3 impregnated silica column. Purification was performed on two 5 g

batches yielding 19 in a minimum purity of 94% according to NMR, and 73% chemical yield.

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With isomerically pure allylic alcohol 19 in hand, the synthesis was continued by means of a Charette asymmetric cyclopropanation. Both enantiomers could be obtained in high enantioselectivity using chiral promotor 33a and 33b to obtain 18a and 18b, respectively. The ee was determined using chiral HPLC by converting 18 into the bisbenzoate ester (Scheme 11). Next, alcohol 18 was protected as a pivaloate ester, and the silyl ether was deprotected using TBAF to furnish alcohol 31a and 31b in excellent yields over three steps. Finally, Appel bromination of the primary alcohol under standard conditions resulted in fragment B in excellent yield for both enantiomers. Starting from alkyne 30, fragment B could be synthesized in 38% yield (average of both enantiomers) and 94% ee in eight steps.

Scheme 11. Ee determination of 18 by the transformation into the corresponding bisbenzoate

esters 32a and 32b and analysis by chiral HPLC.

2.4 Fragment C

The retrosynthesis of fragment C, the most lipophilic fragment containing the syn α-methyl methoxy unit, starts with the disconnection between the methoxy’s α and β carbons revealing epoxide 34 (Scheme 12). This epoxide can undergo nucleophilic addition by the Grignard reagent obtained from chloride 35. Chloride 35 is obtained by a simple substitution of the corresponding commercially available alcohol in one step. Epoxide 34 can in turn be obtained by intramolecular substitution of an in situ generated primary tosylate by its neighboring alkoxide. Since Suzuki-Fu cross-coupling reactions allow for almost any arbitrary disconnection in the fragment, we embarked on also exploiting this feature to our advantage at this stage of the synthesis. Thus, disconnection at the designated position results in primary bromide 36 and 1-hexadecene (4), of which the latter is also used in the Suzuki-Fu cross-coupling in fragment A. This specific disconnection allows the interconversion of bromide 36 to α,β-unsaturated ester 37, which is required for the installment of the methyl branch by applying a highly stereo- and regio-selective conjugate addition of MeLi. Both enantiomers of unsaturated ester 37 are known in the literature, but require a multistep synthesis, either from D-mannitol or L-gulonolactone. Therefore, we attempted to obtain aldehyde 38 directly from enantiopure solketal, of which both enantiomers can be obtained commercially in high ee.

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Scheme 12. Retrosynthesis of fragment C.

2.4.2 Synthesis of 37

Although the oxidation of solketal (39) to 38a (Table 1) seems straightforward on paper, we did not find great support for this reaction in the literature. The number of applications is very limited, but more important, the notions regarding the aldehyde’s stereochemical integrity during reaction and upon storage were conflicting.[19–22]

Realizing that several obstacles had to be overcome: i.e. volatility, water solubility making extractions difficult, and the potential for racemization, we nevertheless attempted the oxidation of solketal (39) to 38a as indicated in Table 1.

The screening of conditions started with a DMP oxidation of 39 to aldehyde 38 (Table 1, entry 1). Because of the product’s volatility we were unable to monitor the progress of the reaction by TLC, and therefore we had to rely on NMR analysis of samples taken from the reaction mixture. Although after 2 h of reaction time NMR showed complete disappearance of alcohol 39, the spectrum gave rise to a complex set of signals between 5 and 3 ppm, which led us to conclude that either during the reaction or workup the product suffered from polymerization.[21]_{The HWE reaction of the obtained crude with}

4 equiv. of triethyl phosphonoacetate (40, Figure 1) in 6 M aqueous K2CO3 did result in

product, which was easily purified by filtration over a plug of silica. However, the yield was low (19% over two steps) and the product consisted of an E/Z mixture in a ratio of approximately 8:1. In order to determine whether the acidic conditions arising from the DMP oxidation caused the degradation (or polymerization), the DMP oxidation was also performed under buffered conditions. Unfortunately, oxidation with DMP in the presence of 4 equiv. of solid NaHCO3 (entry 2) gave after 2 h reaction time a NMR

spectrum that resembled the crude that was obtained without buffering. In order to minimize loss of the volatile product, the extracts of the DMP reaction were only

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partially concentrated and to this crude in DCM was added the reagents for the HWE (entry 2, conditions B). Although product formation seemed to be very minimal on NMR, the use of a two phase system resulted in selective formation of only the desired

E isomer.

Table 1. Conditions screening for the oxidation of solketal to 38 and subsequent olefination.

entry Conditions A Conditions B Yield_(%)a

1 DMP (1.1 eq), DCM, rt, 2 h 40 (4 eq), 6 M K2CO3, rt, 16 h 19b 2 DMP (1.1 eq), NaHCO_{DCM, rt, 2 h} 3 (4 eq) 40 (4 eq), 6M K_{rt, 16 h}2CO3, DCM n.d.

3c _{Swern, DCM, 2 h} _{41, DCM, rt, 16 h} ₉₉d

4c _{Swern, DCM, 2 h} _{40 (4 eq), 6 M K}

2CO3, rt, 16 h 78 5 TEMPO (0.5 mol%), NaOCl _{(1.1 eq)} 40 (4 eq), 6 M K2CO3, rt, 16 h 0.4 a) yield of pure product after column. b) E/Z ratio of 8:1. c) performed as a 1 pot reaction. d) E/Z ratio of 1.4:1

With this gained insight we investigated a one pot two step procedure, in which the intermediate workup was eliminated. Therefore, after Swern oxidation of 38, stabilized Wittig reagent 41 (Figure 1) was added directly to the reaction mixture (entry 3) resulting in a quantitative yield after column, but in a poor E/Z selectivity of 1.4:1. Since HWE reagents generally result in better E/Z selectivity, directly after Swern oxidation of 39 HWE reagent 40 was added along with 6 M K2CO3 (entry 4). After

overnight stirring a satisfying 78% yield was obtained after a quick filtration over silica. More importantly, NMR analysis of the product showed no detectable amounts of the undesired Z-isomer. Unfortunately, comparison of the optical purity of 37 ([α]D = -22.7

(c = 0.57, CHCl3)) with literature did not result in a conclusive answer, since the

reported optical rotations in literature seemed to vary a lot. When chiral HPLC was used it became evident that partial racemization had taken place. Finally, TEMPO oxidation followed by HWE reaction using 40 with intermediate workup resulted in a poor yield of only 0.4%. We therefore decided to abandon this strategy and stick to the reported methodology starting from either mannitol or L-gulonolactone.

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Figure 5. Reagents used for the olefination of 38.

The synthesis of 37b started with the oxidative cleavage of commercially available D

-mannitol bisacetonide 42 using NaIO4,[23] yielding the aldehyde in excellent yield after

careful evaporation of the volatiles (Scheme 13a). The crude aldehyde was then submitted to a HWE reaction resulting in 37b in good yield as a single enantiomer.[24]

Scheme 13. Synthesis of 37 using literature procedures.

In a similar way the enantiomer (37a) was prepared following a slightly modified literature procedure starting from L-gulonolactone (43) (Scheme 13b).[25]_{The sequence}

started with selective acetalisation, followed by NaIO4 oxidation. Then, in the same pot

the HWE reaction was performed by addition of 40 (instead of methyl diisopropoxyphosphinyl acetate, as used in the literature[25]_{). When using only 1 equiv.}

of 40 a low yield of around 25% was obtained. Fortunately, by increasing the equivalency of 40 to 2, the yield could be increased to 55% of 37a over three steps (compared to 34% in literature[25]_{). Minor amounts of the cis isomer were evident on}

TLC, but could be completely separated by column chromatography.

2.4.3 Completion of the fragment

With both enantiomers of the chiral building block 37 in hand, the synthesis continued with a highly regio- and diastereoselective conjugate addition of MeLi,[26]_{resulting in}

the installment of both contiguous stereocenters of fragment C (Scheme 14). Then, reduction of ester 44 to primary alcohol 45 with LiAlH4 followed by Appel reaction

resulted in 36, needed for Suzuki-Fu cross-coupling, in good yields over two steps for both enantiomers. Chain elongation by applying a Suzuki-Fu cross-coupling at this stage of the synthesis was proven not to be straightforward. In situ formation of the active catalyst by the reduction of Pd(OAc)2 with the bulky PCy3 ligand, as reported by

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Complete consumption of the bromide was realized by utilizing 5 mol% of the preformed and commercially available Pd(PCy3)2 catalyst. The catalyst loading could be

reduced to 2.2 mol% when the reaction was performed on a 6.7 g scale of 36, but at the expense of a reaction time of five days.

Scheme 14. Completion of the synthesis of fragment C.

Next, 46 was deprotected with aqueous HCl to afford diol 47. Although diol 47 could be purified by either column chromatography or recrystallization, it was generally of sufficient purity to be used without any form of purification in the epoxidation reaction, yielding 34 in excellent yields over three steps. Epoxide opening of 34 with Grignard reagent 49, obtained from the corresponding chloride, under CuCl catalysis minimized the formation of halohydrin byproducts[27]_{and afforded the secondary alcohol 48.}

Finally, methylation of the hydroxy group with an excess of MeI and NaH resulted in the formation of fragment C in good yield over two steps. Starting from 37, fragment C was obtained in 45% yield (average of both enantiomers) over eight steps.

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2.5 Conclusion

Because the absolute stereochemistry in the cyclopropyl unit and to a lesser extent in the methoxy methyl moiety has not been unambiguously established, we opted for the synthesis of four isomers in which the absolute stereochemistry in the syn methoxy methyl and cis cyclopropyl was varied. Therefore, a convergent synthesis of mycolic Acid from three fragments, each containing one of the three chiral functionalities (i.e. α-alkyl β-hydroxy acid, syn methoxy methyl or cis cyclopropyl) (Scheme 1) seemed to be the most logical approach.

This chapter describes the research that was performed in order to obtain the fragments necessary for the synthesis of mycolic acid. Fragment A was synthesized in nine steps in 24% overall yield starting from commercially available pentadecanolide (6). Key steps are the one pot silylation/hydrogenation/Fukuyama reduction for the synthesis of aldehyde 5, and the Masamune anti aldol reaction for the installment of the stereochemistry in one step, yielding the product in good dr after a laborious chromatographic purification.

The synthesis of fragment B was attempted using three different routes, as the key intermediate 19 proved to be very hard to obtain in isomerically pure form. Our first approach resulted in significant isomerization towards the trans isomer during a DIBAL reduction of 29. An attempt to proceed via the cis vinyl iodide failed as well, since the metalation was unsuccessful. Fortunately, by subjecting propargylic alcohol 20 to a nickel boride reduction, the desired allylic alcohol 19 could be obtained in pure form after purification on a AgNO3 impregnated silica column in order to remove the

over-reduced side product. Fragment B was synthesized in eight steps in a 38% overall yield and 94% ee.

Our attempts to obtain α,β-unsaturated ester 37 via the oxidation of commercially available enantiopure solketal were unsuccessful. Although a very scalable and practical high yielding one pot oxidation/HWE sequence was developed, partial racemization of the stereocenter could not be prevented, limiting this method to racemic synthesis of 37 only. As the reported optical rotations in literature vary considerably, we fear that the racemization of this compound has been occasionally overlooked, and syntheses have been carried out with optically impure material. By applying known chemistry, both enantiomers of 37 were prepared starting from either mannitol or L-gulonolactone. Starting from known 37, fragment C could be obtained as a single isomer in an excellent 45% yield over eight steps, making it the highest yielding fragment in this synthesis. Key steps in the synthesis were a Suzuki-Fu cross-coupling and a regioselective CuCl catalyzed Grignard addition to an epoxide.

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2.6 Possible points of improvement

The presented chemistry in this chapter allowed for the gram-scale synthesis of the required fragments in high optical purity. However, in term of practicality several steps could still benefit from a different or optimized approach. Starting with the Masamune aldol reaction, this reaction afforded the products in a 9:1 dr. Although this selectivity is good, the separation of the diastereomers by column chromatography on silica was problematic. In fact, not a glimpse of separation was observed by TLC. Fortunately, very careful chromatography and 1_{H-NMR analysis of all the fractions containing}

product according to TLC resulted in partial separation of isomers, and an increase of the diastereomeric purity. The product was obtained in reasonable chemical yield (55%, 97:3 dr) after three successive laborious column purifications. TLC using diol-silica coated plates also did not indicate that separation was possible as consistently only one indivisible spot was obtained. It is possible that the use of nitrile, amine or carboxylic acid modified silica results in an improved separation, but since the difference in polarity between the diastereomers is probably “cancelled out” by the long aliphatic tails in the molecule, this approach is most likely deemed to fail.

In 2015 the group of Williams[28]_{reported the asymmetric synthesis of}

corynomycolates. These lipids are similar to mycolic acids in the sense that they contain the same α-alkyl β-hydroxy segment, but lack the cyclopropyl and α-methyl methoxy functionalities and contain significantly shorter lipid tails. For the installment of the sole stereocenters (i.e. the α-alkyl β-hydroxy moiety) the authors used a boron mediated diastereoselective anti aldol reaction of a ketone derived from a chiral lactate, reported by the Paterson[29]_{group in 1994. The aldol reaction of chiral ketone 53 with an}

aldehyde similar to ours resulted in the product as a single isomer in 77% yield after column chromatography. Although the authors did not discuss the obtained diastereomeric ratio in the crude product, it seems from the paper that the formation of the undesired isomer must have been very low, or that the separation was not a major issue, as was the case in our approach. Therefore, the aldol reaction of 57 with aldehyde

5 could be a viable alternative to the Masamune aldol reaction. The carboxylic acid can

be obtained in a straightforward four step procedure consisting of deprotection of the benzoyl group, reduction of the ketone, followed by oxidative cleavage of the obtained 1,2-diol, and a Pinnick oxidation. All in all, it is clear that, although the literature on asymmetric aldol reactions is massive, the toolbox for enantioselective anti aldol reactions is pretty empty.

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Scheme 15. Alternative anti selective aldol reaction applied by Williams et al.

A second point of improvement would be the synthesis of allyl alcohol 19. The AgNO3

impregnated silica column purification after nickel boride reduction of 20 did result in pure material, but also in this case separation was not so trivial and NMR analysis of fractions containing product was necessary in order to determine which fractions to combine. Moreover, a high loading of silver was necessary, resulting in considerable costs. Furthermore, separation of a second batch on the same column resulted in a huge decrease in the resolution, and it was only after reloading the column with more silver nitrate that acceptable separations were obtained again, indicating significant leaching of the silver.

A viable alternative route could possibly proceed via the hydrozirconation of propargylic alcohol 20 (Scheme 16A). Hydrozirconation using Schwartz reagent is known for its high cis hydrometallating selectivity,[30]_{and would therefore form}

zirconium species 59. Since the vinylzirconium intermediate will be trapped with a proton source (such as methanol), and effectively two hydrogens are added to the alkyne, regioselectivity in the hydrometalation is irrelevant making both 59a and 59b a productive intermediate towards 19. Alternatively, if hydrozirconation of 20 does not proceed in high cis selectivity, hydrozirconation of 60, obtained by methylation of 23, could possibly lead to olefin 61. In turn, 61 could be oxidized to 19 by subjecting it to an allylic oxidation with selenium(IV) oxide (Scheme 16B).

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Scheme 16. Synthesis of a cis alkene via hydrozirconation of an alkyne.

In 2014 the group of Williams[31]_{also reported the non-stereoselective cyclopropanation}

of terminal alkynes, resulting in cyclopropenes. The resolution of the cyclopropene enantiomers was possible after derivatization with Evans’ auxiliary[32]_{(Scheme 17). In a}

project like this where both enantiomers are desired, this approach becomes an attractive option. After resolution by conventional column chromatography, which is possible on gram scale, hydrogenation of the cyclopropene resulted in the desired cis cyclopropyl derivative. Although the authors reported that up to 10% trans cyclopropyl isomer was formed, column chromatography enabled separation of the unwanted trans isomer from the desired cis isomer.

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2.7 Experimental section

General remarks

All moisture sensitive reactions were performed using flame-dried glassware under nitrogen atmosphere using standard Schlenk techniques and dry solvents. Reaction temperatures below 0 °C refer to internal temperatures, while reaction temperatures higher than rt refer to heating bath temperatures. Dry solvents were taken from a MBraun solvent purification system (SPS-800). All other reagents were purchased from Sigma-Aldrich, Acros, TCI Europe, Strem chemicals or Fluorochem and used without further purification unless noted otherwise.

TLC analysis was performed with Merck silica gel 60/Kieselguhr F245, 0.25 mm. Compounds were visualized using either a KMnO4 stain (K2CO3 (40 g), KMnO4 (6 g),

water (600 ml) and 10% NaOH (5 ml)), anisaldehyde stain (EtOH (135 ml), H2SO4 (5

ml), AcOH (1.5 ml), p-anisaldehyde (3.7 ml)), PMA stain (phosphomolybdic acid (10 g) in ethanol (100 ml)) or elemental iodine.

Flash chromatography was performed using SiliCycle silica gel type SiliaFlash P60 (230-400 mesh) as obtained from Screening Devices.

1_{H- and}13_{C-NMR spectra were recorded on a Agilent MR400 (400 and 100 MHz,}

respectively) or a Bruker Avance NEO 600 (600 and 150 MHz, respectively). CDCl3

was used as solvent unless stated otherwise. Chemical shift values are reported in ppm with the solvent resonance as the internal standard (CDCl3: δ7.26 for 1H, δ77.16 for 13_{C). Data are reported as follows: chemical shifts, multiplicity (s = singlet, d = doublet,}

dd = double doublet, ddd = double double doublet, dt = double triplte, td = triple doublet, t = triplet, q = quartet, p = pentet, b = broad, m = multiplet), coupling constants

J (Hz), and integration.

Enantiomeric excesses were determined by Chiral HPLC analysis using a Shimadzu LC-10ADVP HPLC instrument equipped with a Shimadzu SPD-M10AVP diode-array detector. Integration was performed at 254 nm and retention times are given in min. High resolution mass spectra (HRMS) were recorded on a Thermo Scientific LTQ Orbitrap XL.

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40

Experimental procedures

2.7.1 Fragment A: Compound 8:

To a cooled solution of lactone 6 (14.0 g, 58.2 mmol) in DCM (290 ml) at ˗80 °C was added DIBAL (1 M in DCM, 64 ml, 64 mmol, 1.1 equiv.) at such a rate that the temperature stayed below -75 °C. When the reaction was complete according to TLC (1.5 h), the reaction was quenched by careful addition of water (2.56 ml), NaOHaq

(15%, 2.56 ml) and water (6.4 ml). The mixture was allowed to reach rt by removal of the cooling bath. After 15 min stirring at rt, anhydrous MgSO4 was added and stirring

was continued for 15 additional min. Then the mixture was filtered and concentrated in

vacuo, yielding the crude lactol 7 (11.2 g, 46.2 mmol,) as a white solid.

The obtained lactol was submitted into the HWE reaction in two batches of 5.2 g:

To a solution of the crude lactol 7 (5.2 g, 23 mmol) in toluene (80 ml) at 75 °C was added the ylide[4]_{, followed by sufficient THF to make the reaction completely}

homogeneous (around 40 ml). After overnight stirring, TLC indicated complete consumption of the lactol. The reaction mixture was allowed to cool to rt, and absorbed on celite. Purification by flash column chromatography using pentane/ether (6:4) yielded a yellow solid (13.39 g, 40.77 mmol, 70% combined yield, over two steps).

1_{H-NMR (400 MHz, CDCl} 3) δ 6.89 (dt, J = 14.6, 6.9 Hz, 1H), 6.09 (d, J = 15.5 Hz, 1H), 3.64 (t, J = 6.6 Hz, 2H), 2.94 (q, J = 7.5 Hz, 2H), 2.18 (q, J = 7.3 Hz, 2H), 1.56 (p, J = 6.8 Hz, 2H), 1.49 – 1.19 (m, 26H). 13_{C-NMR (101 MHz, CDCl} 3) δ 190.31, 145.55, 128.71, 63.02, 32.86, 32.24, 29.70, 29.67, 29.57, 29.52, 29.44, 29.23, 28.06, 25.84, 23.09, 14.89. HRMS (ESI) Calcd. for C19H36NaO2S ([M + Na]+): 351.233, found:

351.234.

Compound 5:

Thioester 8 (1.0 g, 3.0 mmol) was dissolved in acetone (30 ml) and cooled to 0 °C using an ice-water bath, and Pd/C (10 weight%, 161 mg, 0.15 mmol, 5 mol%) was added in

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41

one portion. Triethylsilane (1.94 ml, 12.1 mmol, 4 equiv.) was added over 15 min, then the cooling bath was removed, and the reaction was allowed to stir for 5 h at rt. Then, celite was added, and the volatiles were removed. Flash column purification using 2-4% ether in pentane yielded a colorless liquid (0.95 g, 2.46 mmol, 82% yield).

Note: the reaction could be scaled up to a 12 gram scale with a reduced yield of approximately 60% 1_{H-NMR (400 MHz, CDCl} 3) δ 9.76 (t, J = 1.9 Hz, 1H), 3.59 (t, J = 6.7 Hz, 2H), 2.41 (td, J = 7.4, 1.8 Hz, 2H), 1.62 (q, J = 7.2 Hz, 2H), 1.56 – 1.48 (m, 2H), 1.37 – 1.21 (m, 24H), 0.96 (t, J = 7.9 Hz, 9H), 0.59 (q, J = 8.0 Hz, 6H). 13_{C-NMR (101 MHz, CDCl} 3) δ 202.97, 63.08, 44.00, 33.03, 29.77, 29.75, 29.72, 29.69, 29.58, 29.54, 29.46, 29.27, 25.93, 22.19, 6.85, 6.67, 5.91, 4.53. HRMS (ESI) Calcd. for C23H48NaO2Si ([M +

Na]+_{): 407.332, found: 407.333.} Compound 13:

To a solution, cooled at 0 °C, of oxalyl bromide (2 M in DCM, 8.9 ml, 17.8 mmol, 1.4 equiv.) was added 10-bromodecanoic acid (3) (4.47 g, 17.8 mmol, 1.4 equiv.) in small portions. After complete addition, the RM was allowed to warm up to rt and stirred at this temperature until gas formation ceased (around 1 h). Quenching of a small sample with methanol and elution on TLC (pentane /ethyl acetate 8:2) showed complete conversion into the methyl ester, and thus complete conversion of the carboxylic acid into the acid bromide.

In a separate Schlenk flask, 2 (5.38 g, 12.7 mmol, 1.0 equiv.) was dissolved in DCM (60 ml), followed by the addition of pyridine (3.02 g, 38.1 mmol, 3.1 ml, 3.0 equiv.) and the mixture was cooled to 0 °C using an ice-water bath. Then, the freshly made acid bromide solution was carefully added after which the reaction was allowed to stir for 2 h at rt. After TLC confirmed complete consumption of 2, the reaction mixture was diluted with DCM (25 ml) and washed with saturated aqueous NaHCO3 (100 ml). The

aqueous layer was back extracted with DCM (2× 75 ml) and the combined organic layer was washed with 1 M HClaq (150 ml), water (150 ml), brine (150 ml), dried over

MgSO4 and concentrated in vacuo. The crude was purified by flash column

chromatography with 5-10% ethyl acetate in pentane to yield a viscous colorless oil (7.87 g, 12.0 mmol, 94% yield). 1_{H-NMR (400 MHz, CDCl} 3) δ 7.32 (d, J = 7.0 Hz, 2H), 7.28 – 7.22 (m, 2H), 7.22 – 7.15 (m, 4H), 6.95 – 6.80 (m, 4H), 5.83 (d, J = 4.0 Hz, 1H), 4.73 (d, J = 16.6 Hz, 1H), 4.59 (d, J = 16.6 Hz, 1H), 4.10 – 3.99 (m, 1H), 3.39 (t, J = 6.8 Hz, 2H), 2.51 (s, 6H), 2.28 (s, 3H), 2.23 – 2.05 (m, 2H), 1.83 (p, J = 6.9 Hz, 2H), 1.50 (p, J = 7.5 Hz, 2H),

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42

1.40 (p, J = 7.0 Hz, 2H), 1.33 – 1.17 (m, 8H), 1.13 (d, J = 6.9 Hz, 3H). 13_{C-NMR (101}

MHz, CDCl3) δ 171.95, 142.50, 140.16, 138.69, 138.60, 133.37, 132.15, 128.37,

128.34, 127.80, 127.37, 127.08, 125.94, 77.93, 56.71, 48.15, 34.12, 34.01, 32.76, 29.18, 29.09, 28.97, 28.63, 28.08, 24.61, 22.99, 20.88, 12.85. HRMS (ESI) Calcd. for C35H46BrNNaO4S ([M + Na]+): 678.223 and 680.221, found: 678.223 and 680.221. Compound 14:

To a solution of 13 (10.78 g, 16.44 mmol) and triethylamine (5.5 ml, 2.4 equiv.) in DCM (50 ml) at ˗80 °C was added a solution of the boron triflate (12.22 g, 36.35 mmol, 2.2 equiv.) in DCM (30 ml) via syringe pump over 2 h (maintaining an internal temperature of ˗78 °C). After complete addition, enol formation was allowed to take place by stirring for an additional 5 h at this temperature. Then, a solution of aldehyde 5 (7.07 g, 18.4 mmol, 1.1 equiv.) in DCM (20 ml) was added slowly via syringe pump at a rate of 24 ml/h, and the reaction was stirred overnight at ˗78 °C. The reaction was quenched at ˗78 °C by addition of phosphate buffer (pH 7, 65 ml), MeOH (160 ml) and aqueous H2O2 (30%, 20 ml), allowed to reach rt, and stirred for another 24 h. Brine (300

ml) was added and the mixture was extracted with ether (4× 100 ml). The combined organic layer was washed with brine (200 ml), dried over MgSO4 and concentrated in

vacuo yielding 17.33 g as a yellowish thick oil.

The crude was dissolved in THF (100 ml), 2 M HClaq (20 ml) was added and the

mixture was stirred at rt for 1 h. The mixture was neutralized by the addition of saturated aqueous NaHCO3 (200 ml) and extracted with ether (3× 150 ml). The

combined organic layer was dried over MgSO4 and concentrated in vacuo. Careful flash

column chromatography applying 40% ether in pentane during 6 column volumes followed by 45% ether in pentane yielded recovered 13 (2.0 g, 3.05 mmol) and 14 (8.29 g, 8.94 mmol, 55% yield, 67% yield brsm, dr > 97:3)

Note: Because the diastereomers separate very poorly on TLC, all fractions with a dr > 95:5 as judged by NMR were combined. Fractions with dr < 95:5 were combined, concentrated in vacuo, and re-purified using the same conditions. The reported yield is the result of three successive columns.

1_{H NMR (400 MHz, CDCl}

3) δ 7.31 – 7.17 (m, 6H), 7.13 (dd, J = 8.2, 6.7 Hz, 2H), 6.87

– 6.80 (m, 4H), 5.82 (d, J = 5.7 Hz, 1H), 4.75 (d, J = 16.3 Hz, 1H), 4.50 (d, J = 16.4 Hz, 1H), 4.16 (p, J = 6.8, 6.2 Hz, 1H), 3.63 (t, J = 6.7 Hz, 3H), 3.38 (t, J = 6.8 Hz, 2H), 2.42 (s, 7H), 2.36 (d, J = 7.4 Hz, 1H), 2.28 (s, 3H), 1.80 (dt, J = 14.8, 6.9 Hz, 2H), 1.60 –

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43 0.93 (m, 42H). 13_{C NMR (101 MHz, CDCl} 3) δ 174.64, 142.51, 140.33, 138.39, 137.94, 133.15, 132.10, 128.31, 128.22, 128.06, 127.25, 126.69, 77.97, 77.36, 72.29, 62.90, 56.40, 51.31, 48.09, 35.30, 33.97, 32.78, 32.74, 29.68, 29.66, 29.64, 29.62, 29.59, 29.47, 29.34, 29.07, 28.51, 28.07, 27.07, 25.79, 25.59, 22.89, 20.91, 14.44. HRMS

(ESI) Calcd. for C52H80BrNNaO6S ([M + Na]+): 948.479 and 950.477, found: 948.479

and 950.478.

Compound 15:

To a solution of 14 cooled at 0 °C (8.30 g, 8.95 mmol, 1.0 equiv.) and 2,6-lutidine (5.76 g, 53.71 mmol, 6.2 ml, 6.0 equiv.) in DCM (30 ml) was added TBSOTf (9.47 g, 35.8 mmol, 4.0 equiv.). After complete addition, the cooling bath was removed and the reaction mixture was allowed to stir at rt for 3 h. Then, the reaction mixture was diluted with DCM (50 ml) and washed with 0.5 M HClaq (300 ml). The layers were separated

and the aqueous layer was extracted with DCM (3× 75 ml). The combined organic layer was washed with brine (150 ml), dried over MgSO4 and concentrated in vacuo, which

yielded an orange oil. The crude was purified by column chromatography using 6% ether in pentane yielding a yellowish oil (9.68 g, 8.38 mmol, 94% yield).

1_{H-NMR (400 MHz, CDCl} 3) δ 7.35 – 7.31 (m, 2H), 7.26 – 7.16 (m, 4H), 7.12 – 7.06 (m, 2H), 6.85 – 6.78 (m, 4H), 5.72 (d, J = 6.2 Hz, 1H), 4.80 (d, J = 16.2 Hz, 1H), 4.42 (d, J = 16.2 Hz, 1H), 4.16 (p, J = 6.7 Hz, 1H), 3.91 – 3.83 (m, 1H), 3.60 (t, J = 6.6 Hz, 2H), 3.38 (t, J = 6.8 Hz, 2H), 2.52 – 2.45 (m, 1H), 2.39 (s, 6H), 2.29 (s, 3H), 1.85 – 1.76 (m, 2H), 1.55 – 1.42 (m, 4H), 1.40 – 1.06 (m, 39H), 0.96 (t, J = 7.1 Hz, 2H), 0.90 (s, 9H), 0.87 (s, 9H), 0.07 – 0.02 (m, 12H).13_{C-NMR (101 MHz, CDCl} 3) δ 172.65, 142.41, 140.40, 138.28, 133.18, 132.13, 128.38, 128.27, 128.16, 127.91, 127.37, 126.87, 77.82, 77.36, 72.77, 63.34, 56.47, 51.69, 48.22, 33.86, 33.56, 32.96, 32.82, 29.76, 29.75, 29.72, 29.70, 29.65, 29.63, 29.52, 29.12, 28.60, 28.12, 27.63, 26.91, 26.07, 25.96, 25.88, 25.01, 22.91, 20.94, 18.41, 18.13, 15.03, -4.31, -4.53, -5.17. HRMS (ESI) Calcd. for C64H108BrNNaO6SSi2 ([M + Na]+): 1176.65 and 1178.65, found: 1176.65 (100%)

and 1178.65 (100%).

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Hydroboration:

To a solution of 1-hexadecene (4) (2.73 g, 3.5 ml, 12.2 mmol, 1.0 equiv.) in THF (6 ml) was added 9-BBN dimer (1.73 g, 0.58 equiv.) and the resulting solution was stirred overnight at rt. NMR indicated complete conversion of 4 (no detectable olefin signals). Cross-coupling:

To the preformed solution of the alkylborane (1.5 equiv.) was added KH2PO4·H2O (2.87

g, 12.5 mmol, 1.5 equiv.) followed by Pd(PCy3)2 (425 mg, 0.64 mmol, 7.7 mol%), and

the reaction mixture was stirred for 30 min at rt, which resulted in a clear yellow solution. Then, 15 (9.59 g, 8.30 mmol, 1.0 equiv.), as a solution in THF (18 ml), was added and the reaction mixture was stirred at rt overnight. The reaction mixture was absorbed on celite and purified by flash column chromatography using 1-4% ether in pentane yielding 17 as a colorless oil (9.45 g, 7.26 mmol, 87% yield).

Note: the product contained some apolar impurities which, although on TLC separable, co-eluted with the product during column purification. The product after 1 column purification was used as such in the next reaction since the impurities did not interfere with the reaction.

Compound 17:

HF Stock solution:

Commercially available HF·pyridine (~70 % HF, 4.85 ml) was diluted with anhydrous THF (24 ml) and cooled to 0 °C. Then, anhydrous pyridine (9.7 ml) was added and stirred briefly at this temperature before it was used without further manipulation. Selective deprotection:

Bis-TBS ether 17 (3.81 g, 2.93 mmol, 1.0 equiv.) was dissolved in THF (18 ml) and cooled down to 0 °C using an ice-water bath. The previously formed HF stock solution (11 ml) was added, and the mixture was allowed to reach rt by removing the ice-water bath. The reaction was monitored closely by TLC, and quenched as soon as full conversion was reached (around 3.5 h) by careful addition of saturated aqueous NaHCO3 solution (150 ml, gas formation!) while cooling with an ice-water bath to 0 °C.

The mixture was extracted with DCM (4× 30 ml), and the combined organic layer was washed with, 1M HClaq (75 ml), brine (75 ml), dried over MgSO4 and concentrated in

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chromatography applying a 30-60% ether in pentane gradient, which yielded a yellow oil (3.30 g, 2.78 mmol, 95% yield).

1_{H-NMR (400 MHz, CDCl} 3) δ 7.36 – 7.30 (m, 2H), 7.25 – 7.14 (m, 4H), 7.12 – 7.05 (m, 2H), 6.85 – 6.77 (m, 4H), 5.70 (d, J = 6.2 Hz, 1H), 4.80 (d, J = 16.2 Hz, 1H), 4.42 (d, J = 16.2 Hz, 1H), 4.15 (p, J = 6.8 Hz, 1H), 3.86 (t, J = 6.2 Hz, 1H), 3.64 (q, J = 6.5 Hz, 2H), 2.48 (dt, J = 9.4, 4.9 Hz, 1H), 2.39 (s, 6H), 2.28 (s, 3H), 1.62 – 1.51 (m, 2H), 1.54 – 1.41 (m, 2H), 1.39 – 1.03 (m, 74H), 1.01 – 0.92 (m, 2H), 0.92 – 0.82 (m, 12H), 0.04 (s, 3H), 0.03 (s, 3H). 13_{C-NMR (101 MHz, CDCl} 3) δ 172.81, 142.50, 140.50, 138.36, 138.32, 133.22, 132.20, 128.44, 128.32, 128.21, 127.95, 127.42, 126.91, 77.92, 72.84, 63.14, 56.55, 51.74, 48.29, 33.63, 32.94, 32.06, 29.84, 29.83, 29.81, 29.80, 29.79, 29.76, 29.74, 29.72, 29.70, 29.65, 29.58, 29.49, 29.47, 27.78, 27.02, 26.01, 25.88, 25.09, 22.97, 22.81, 20.99, 18.19, 15.04, 14.24, -4.27, -4.49. HRMS (ESI) Calcd. for C74H127NNaO6SSi ([M + Na]+): 1208.91, found: 1208.90.

Compound 1:

Alcohol 17 (5.53 g, 4.66 mmol 1.0 equiv.) was dissolved in DCM (30 ml) and the resulting solution was cooled to 0 °C using an ice-water bath. Then, Dess-Martin periodinane (2.53 g, 5.97 mmol, 1.3 equiv.) was added, and the reaction was stirred at rt. After 2.5 h, TLC (40% ether in pentane) still indicated the presence of small amounts of starting material. Additional DMP reagent (500 mg, 1.18 mmol, 0.25 equiv.) was added, and the reaction was allowed to stir for 1h at rt, after which TLC indicated full consumption of 17. The reaction mixture was concentrated in vacuo, redissolved in pentane (250 ml) and reagents were washed out with acetonitrile (3× 150 ml). The combined acetonitrile layers were back extracted with pentane (100 ml), and the combined pentane layers were washed with saturated aqueous NaHCO3 solution (200

ml), brine (200 ml), dried over MgSO4 and concentrated in vacuo yielding a yellowish

oil. Flash column chromatography using a 10-20 % ether in pentane gradient yielded 1 as a colorless oil (5.30 g, 4.47 mmol, 96% yield).

1_{H-NMR (400 MHz, CDCl} 3) δ 9.74 (t, J = 2.1 Hz, 1H), 7.38 – 7.32 (m, 2H), 7.27 – 7.19 (m, 3H), 7.19 – 7.14 (m, 1H), 7.13 – 7.04 (m, 2H), 6.85 – 6.79 (m, 4H), 5.73 (d, J = 6.2 Hz, 1H), 4.82 (d, J = 16.2 Hz, 1H), 4.44 (d, J = 16.2 Hz, 1H), 4.16 (p, J = 6.7 Hz, 1H), 3.88 (td, J = 6.1, 3.3 Hz, 1H), 2.50 (dt, J = 9.5, 4.9 Hz, 1H), 2.45 – 2.34 (m, 8H), 2.28 (s, 3H), 1.68 – 1.57 (m, 2H), 1.56 – 1.04 (m, 73H), 1.02 – 0.93 (m, 2H), 0.92 – 0.84 (m, 12H), 0.05 (d, J = 3.1 Hz, 6H). 13_{C-NMR (101 MHz, CDCl} 3) δ 202.92, 172.80, 142.51, 140.50, 138.38, 138.33, 133.23, 132.20, 128.45, 128.31, 128.22, 127.95, 127.42,

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126.90, 77.93, 72.84, 56.56, 51.73, 48.30, 44.04, 33.63, 32.06, 29.85, 29.84, 29.81, 29.80, 29.78, 29.76, 29.74, 29.73, 29.71, 29.66, 29.57, 29.50, 29.48, 29.30, 27.79, 27.01, 26.01, 25.10, 22.98, 22.82, 22.21, 21.00, 18.20, 15.03, 14.25, -4.26, -4.49.

HRMS (ESI) Calcd. for C74H125NNaO6SSi ([M + NH4]+): 1201.93, found: 1201.93. 2.7.2 Fragment B:

Compound 23:

Alkyne zipper reaction:

NaH (60% dispersion in mineral oil, 16 g, 400 mmol, 3.65 equiv.) was washed with pentane (3x 50 ml) and 1,3-diaminopropane (182 ml) was added to the white solid residue. The resulting mixture was warmed to 70 °C and stirred at this temperature for 30 min (reaction turned into a dark brown solution). Then, the reaction mixture was cooled to 0 °C with an ice-water bath after which alcohol 30 (12.28 g, 109.5 mmol, 1.0 equiv.) was added neat via syringe. Upon complete addition of the alcohol, the ice-water bath was removed, and the reaction was stirred at rt for 1.5 h. Then, the reaction was cooled to 0 °C and quenched with ice-water (300 ml). The solution was extracted with ether (5× 75 ml), and the combined organic layer was washed with 2 M HClaq (3× 200

ml), water (1× 200 ml), (brine 1× 200 ml), dried over MgSO4 and concentrated in vacuo

to yield a yellow oil (8.55 g, 76.2 mmol, 70%). The water layers were back extracted with ether to provide additional material as a dark orange oil (2.58 g, 23 mmol, 21% yield).

TBDPS protection:

A solution of the crude alcohol (8.55 g, 76.2, 1.0 equiv.) in DCM (170 ml) was cooled to 0 °C using an ice-water bath, followed by the addition of imidazole (10.8 g, 158 mmol, 2.1 equiv.). After a brief stirring at 0 °C, TBDPSCl (25.14 g, 91.47 mmol, 23.8 ml, 1.2 equiv.) was added via syringe over 10 min, and the reaction mixture was stirred at 0 °C for 15 min after which it was allowed to warm up to rt overnight by removing the ice-water bath. The reaction was quenched with water (200 ml), and the layers were separated. The water layer was extracted with DCM (3× 75 ml). The combined organic layer was washed with brine (100 ml), dried over MgSO4 and concentrated in vacuo

yielding a yellow oil. The crude was purified by flash column chromatography by eluting with pure pentane for roughly 2 column volumes, after which a 1% ether in pentane mobile phase was applied, yielding a colorless oil (25.15 g, 71.75 mmol, 94% yield, 86% yield over two steps).

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47 1_{H-NMR (400 MHz, CDCl} 3) δ 7.71 – 7.63 (m, 4H), 7.48 – 7.33 (m, 6H), 3.67 (t, J = 6.3 Hz, 2H), 2.17 (tdd, J = 6.8, 2.6, 1.2 Hz, 2H), 1.93 (td, J = 2.6, 1.2 Hz, 1H), 1.63 – 1.42 (m, 6H), 1.05 (s, 9H). 13_{C-NMR (101 MHz, CDCl} 3) δ 135.70, 134.20, 129.65, 127.73,

84.67, 68.34, 63.84, 32.15, 28.35, 27.02, 25.12, 19.36, 18.53. HRMS (ESI) Calcd. for C23H30NaOSi ([M + Na]+): 373.196, found: 373.196.

Compound 20:

To a solution of 23 (25.10 g, 71.60 mmol, 1.0 equiv.) in THF (250 ml) at 0 °C was added BuLi (1.6 M in hexane, 82 ml, 131 mmol, 1.8 equiv.) dropwise, and the mixture was stirred for 30 min at 0 °C. Paraformaldehyde (11.3 g, 358 mmol, 5.0 equiv.) was added in portions and stirring was continued at 0 °C for 30 min. The ice-water bath was removed and the reaction was allowed to stir overnight at rt. The reaction was quenched with 1:1 saturated aqueous NH4Cl/water (100 ml) and the THF was removed in vacuo.

The aqueous residue was extracted with ether (3× 100 ml) and the combined organic layer was washed with brine (1× 200 ml), dried over MgSO4 and concentrated in vacuo.

The crude was purified by flash column chromatography using a 15-40% ether in pentane gradient yielding a colorless oil (19.15 g, 50.33 mmol, 70% yield).

1_{H-NMR (400 MHz, CDCl}

3) δ 7.70 – 7.63 (m, 4H), 7.45 – 7.32 (m, 6H), 4.29 – 4.17

(m, 2H), 3.66 (t, J = 6.4 Hz, 2H), 2.24 – 2.16 (m, 2H), 1.61 – 1.39 (m, 7H), 1.05 (s, 9H).

13_{C-NMR (101 MHz, CDCl}

3) δ 135.70, 134.20, 129.66, 127.72, 86.58, 78.54, 63.85,

51.54, 32.16, 28.43, 27.01, 25.19, 19.36, 18.85. HRMS (ESI) Calcd. for C24H32NaO2Si

([M + Na]+_{): 403.206, found: 403.206.} Compound 19:

NaBH4 (1.18 g, 31.3 mmol, 1.0 equiv.) was added portionwise to a stirring solution of

Ni(OAc)2·4H2O (7.78 g, 31.3 mmol, 1.0 equiv.) in absolute ethanol (150 ml) under a

hydrogen blanket (1 atm) at rt, forming a black heterogeneous suspension. After 15 min stirring, ethylenediamine (3.76 g, 4.2 ml, 62.5 mmol, 2.0 equiv.) was added followed by

20 (11.90 g, 31.27 mmol, 1.0 equiv.) dissolved in EtOH (40 ml) while maintaining the

hydrogen blanket (1 atm). TLC (4% ether in toluene, the TLC was ran twice) showed complete conversion after 20 min, after which the reaction mixture was filtered over a plug of silica. Eluting was continued with ether until TLC indicated complete recovery of the product, yielding a colorless oil (11.38 g). The crude was purified by column

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48

chromatography in two batches over AgNO3 impregnated (20 wt%) silicagel (250 g),

which was prepared by eluting a concentrated AgNO3 solution in acetonitrile (50 g

AgNO3 dissolved in a minimal amount of acetonitrile at rt) over a dry silica column

covered with aluminium household foil in order to exclude light. The column was dried by blowing compressed air through the column for 2h. Then, the column was flushed with 30% ether in pentane, the crude was loaded on the column and elution was continued with 30% ether in pentane. When most of the product had eluted from the column, the last traces of product were removed by flushing with 100% ether. All fractions with purity > 94% according to 1_{H-NMR were combined and evaporated}

(integral of the olefin hydrogen signal higher than 0.94 with respect to the aliphatic signal was used as a guideline for the desired purity. Spectra were recorded while applying a relaxation time of 10 sec to allow for accurate integration.). Before purifying the second batch, the column was reloaded with an additional 35 gr AgNO3 by repeating

the aforementioned process. Purification over two batches yielded a colorless oil which was dissolved in ether (500 ml) and washed with 12% aqueous ammonia (3× 75 ml) in order to remove any leached Ag. The combined organic layer was washed with water (2× 200 ml), dried over MgSO4 and concentrated in vacuo to yield a colorless oil (8.70

g, 22.7 mmol, 73% yield). 1_{H-NMR (600 MHz, CDCl} 3) δ 7.62 – 7.54 (m, 4H), 7.38 – 7.26 (m, 6H), 5.58 – 5.47 (m, 1H), 5.47 – 5.39 (m, 1H), 4.11 (d, J = 6.0 Hz, 2H), 3.58 (t, J = 6.5 Hz, 2H), 2.03 – 1.94 (m, 2H), 1.53 – 1.44 (m, 2H), 1.33 – 1.25 (m, 4H), 1.10 (br. s, 1H), 0.98 (s, 9H). 13_{C-NMR (151 MHz, CDCl} 3) δ 135.67, 134.22, 133.01, 129.62, 128.62, 127.69, 63.98,

58.66, 32.48, 29.42, 27.49, 27.01, 25.50, 19.34. HRMS (ESI) Calcd. for C24H34NaO2Si

([M + Na]+_{): 405.222, found: 405.222.} Compound 18a:

A 3-neck flask was connected to a vacuum/nitrogen line, a thermometer, and the remaining neck was stoppered with a septum. The flask was charged with DCM (60 ml) followed by 1,2-dimethoxyethane (2.13 g, 5.5 ml, 23.6 mmol, 2.0 equiv.).[8] _The

resulting solution was cooled to ˗10 °C, followed by the addition of diethylzinc (2.91 g, 2.4 ml, 23.6 mmol, 2.0 equiv.). Then, diiodomethane (12.6 g, 3.8 ml, 47.2 mmol, 4.0 equiv.) was added while keeping the internal temperature between ˗16 °C and ˗13

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49

°C. After complete addition of the diiodomethane the reaction was left at ˗15 °C for 15 min. The ligand 33a (3.82 g, 14.2 mmol, 1.2 equiv.) as a solution in DCM (10 ml) was added while keeping the internal temperature below ˗10 °C. Immediately after complete addition of the ligand, a preformed solution of alcohol 19 (4.51 g, 11.8 mmol, 1.0 equiv.) in DCM (10 ml) was added and the reaction mixture was allowed to stir for 15 min at ˗15 °C. After 15 min, the cooling bath was removed and the reaction was stirred overnight at rt. The reaction was quenched by addition of saturated aqueous NH4Cl (15

ml) followed by 2 M HClaq (75 ml). The mixture was diluted with ether (100 ml) and

transferred to a separatory funnel. The reaction flask was rinsed with ether (25 ml) and 2 M HClaq (20 ml), and both solutions were transferred to the separatory funnel. The

layers were separated, and the aqueous layer was extracted with ether (2× 20 ml). The combined organic layer was transferred to an Erlenmeyer flask, and a solution containing 2 M NaOHaq (80 ml) and aqueous 30% H2O2 (10 ml) was added in one

portion. The resulting biphasic solution was stirred vigorously for 5 min. The layers were separated and the organic layer was washed successively with 2 M HClaq (80 ml),

aqueous saturated sodium sulfite (80 ml), aqueous saturated sodium bicarbonate (80 ml) and brine (80 ml). The organic layer was dried over MgSO4 and concentrated in vacuo

yielding a colorless oil. The crude was purified by flash column chromatography with 30% ether in pentane yielding a colorless oil (5.11 g, 12.9 mmol, 109% yield).

1_{H-NMR (400 MHz, CDCl} 3) δ 7.69 (dt, J = 7.9, 1.9 Hz, 4H), 7.52 – 7.32 (m, 6H), 3.71 – 3.54 (m, 4H), 1.63 – 1.52 (m, 2H), 1.46 – 1.16 (m, 7H), 1.06 (s, 9H), 0.97 – 0.81 (m, 2H), 0.75 – 0.67 (m, 1H), -0.04 (q, J = 5.4 Hz, 1H). 13_{C-NMR (101 MHz, CDCl} 3) δ 135.68, 134.26, 129.60, 127.68, 64.05, 63.41, 32.67, 30.00, 28.63, 27.00, 25.81, 19.35, 18.24, 16.21, 9.62. HRMS (ESI) Calcd. for C23H127NNaO6SSi ([M + Na]+): 405.222,

found: 405.222. HRMS (ESI) Calcd. for C25H36NaO2Si ([M + Na]+): 419.238, found:

419.237.

Note: the purified material still contained n-BuOH.

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The aforementioned procedure using 19 (2.34 g, 6.13 mmol, 1.0 equiv.) and ligand 33b (2.03 g, 7.52 mmol, 1.2 equiv.) resulted in a colorless oil (2.49 g, 6.29 mmol, 103% yield) after flash column chromatography purification.

1_{H-NMR (400 MHz, CDCl} 3) δ 7.67 (d, J = 7.0 Hz, 4H), 7.45 – 7.34 (m, 6H), 3.69 – 3.58 (m, 4H), 1.62 – 1.53 (m, 2H), 1.49 – 1.26 (m, 4H), 1.25 – 1.14 (m, 2H), 1.14 – 1.06 (m, 1H), 1.05 (s, 9H), 0.95 – 0.79 (m, 2H), 0.70 (td, J = 8.3, 4.5 Hz, 1H), -0.05 (q, J = 5.2 Hz, 1H). 13_{C-NMR (101 MHz, CDCl} 3) δ 135.65, 134.22, 129.57, 127.66, 64.03, 63.31, 32.64, 29.97, 28.60, 26.98, 25.78, 19.31, 18.19, 16.17, 9.60. HRMS (ESI) Calcd. for C25H36NaO2Si ([M + Na]+): 419.238, found: 419.237.

Note: the purified material still contained n-BuOH.

Ee determination:

Compound 32a:

Alcohol 18a (135 mg, 0.34 mmol, 1 equiv.) was dissolved in 1 M TBAF in THF (1.0 ml, 1.02 mmol, 3.0 equiv.) and the resulting mixture was stirred overnight. The reaction mixture was concentrated in vacuo and purified by flash column chromatography in 100% EtOAc, which yielded the product as a colorless oil (54 mg, 0.34 mmol, quantitative yield).

The obtained diol (28 mg, 0.18 mmol, 1.0 equiv.) was dissolved in DCM (0.3 ml), and pyridine (114 μl, 1.42 mmol, 8.0 equiv.) and benzoyl chloride (82 μl, 0.71 mmol, 4.0 equiv.) were subsequently added. After 1 h stirring at rt, TLC showed complete conversion, and dimethyl 3-amino propylamine (67 μl, 0.5 mmol, 3.0 equiv.) was added in order to quench the excess benzoyl chloride. After 1 h of stirring at rt, DCM (10 ml) was added to the reaction mixture and the organic layer was washed successively with 1M HClaq (5 ml), saturated aqueous NaHCO3 solution (5 ml), brine (5 ml), dried over

MgSO4 and concentrated in vacuo to yield the product (60 mg, 0.16 mmol, 93% yield)

as a pale yellow oil in sufficient purity according to 1_H-NMR.

1_{H-NMR (400 MHz, CDCl} 3) δ 8.10 – 8.01 (m, 4H), 7.59 – 7.52 (m, 2H), 7.46 – 7.40 (m, 4H), 4.50 (dd, J = 11.7, 6.8 Hz, 1H), 4.30 (t, J = 6.6 Hz, 2H), 4.15 (dd, J = 11.7, 8.9 Hz, 1H), 1.81 – 1.70 (m, 2H), 1.56 – 1.44 (m, 5H), 1.36 – 1.22 (m, 2H), 0.99 – 0.89 (m, 1H), 0.81 (td, J = 8.4, 4.8 Hz, 1H), 0.12 (q, J = 5.3 Hz, 1H). 13_{C-NMR (101 MHz,} CDCl3) δ 166.83, 166.76, 132.92, 132.91, 130.65, 130.62, 129.68, 129.64, 128.44, 128.43, 65.98, 65.12, 29.81, 28.89, 28.69, 26.07, 16.38, 14.41, 10.03. HRMS (APCI) Calcd. for C23H26NaO4 ([M + Na]+): 389.172, found: 389.172.